Cytoplasmic inhibition of gene expression and expression of a foreign protein in a monocot plant by a plant viral vector

Abstract

The present invention relates to recombinant viral vectors encoding a transcriptional unit, that encodes a fusion protein, or a foreign protein or a gene of interest to be silenced, which can be expressed in a host. The present invention also relates to the use of these recombinant viral vectors to express a fusion protein, a foreign protein, to silence a gene of interest in a host. The present invention also relates to the use of these recombinant viral vectors to screen a CDNA or genomic library in order to correlate a nucleotide sequence with a phenotypic or biochemical change.

What is claimed is:  
     
       1. A polynucleotide encoding a promoter operatively linked to a transcription unit, wherein the promoter comprises a promoter functional in a plant or plant cell; and wherein the transcription unit encodes a fusion protein comprising: 
       (a) a viral protein obtained from a plant single-stranded RNA virus, wherein the plant single-stranded RNA virus is a barley stripe mosaic virus (BSMV),  
       (b) a protein of interest, and  
       (c) an autoproteolytic peptide comprising no more than 20 amino acids, wherein the autoproteolytic peptide comprises a 2A autoproteolytic peptide from a foot and mouth disease virus (FMDV), wherein (c) is fused between (a) and (b).  
     
     
       2. The polynucleotide according to  claim 1 , where in the autoproteolytic peptide comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
       3. The polynucleotide according to  claim 1 , wherein the fusion protein comprises no more than one viral protein. 
     
     
       4. The polynucleotide of according to  claim 1 , wherein the viral protein is a BSMV gamma-b protein. 
     
     
       5. The polynucleotide according to  claim 1 , wherein the autoproteolytic peptide is fused to the C-terminus of the viral protein. 
     
     
       6. The polynucleotide according to  claim 1 , wherein the autoproteolytic peptide is fused to the N-terminus of the viral protein. 
     
     
       7. The polynucleotide of according to  claim 1 , wherein the viral protein is a BSMV beta-b protein. 
     
     
       8. The polynucleotide according to  claim 7 , wherein the autoproteolytic peptide is fused to the N-terminus of the viral protein, and where the protein of interest is fused to the N-terminus of the autoproteolytic peptide. 
     
     
       9. The polynucleotide according to  claim 1 , wherein the protein of interest is a plant protein. 
     
     
       10. The polynucleotide according to  claim 9 , wherein the plant protein is a structural protein, enzyme, or a protein involved with pigmentation. 
     
     
       11. A recombinant viral nucleic acid comprising the polynucleotide according to  claim 8 . 
     
     
       12. A plant or plant cell containing the recombinant viral nucleic acid according to  claim 11 . 
     
     
       13. A recombinant virus comprising the recombinant viral nucleic acid according to  claim 11 , wherein the recombinant virus is capable of systemic expression of the fusion protein. 
     
     
       14. A plant or a plant cell infected with a recombinant virus according to  claim 13 . 
     
     
       15. The polynucleotide according to  claim 1 , wherein the fusion protein can be expressed in a plant or a plant cell. 
     
     
       16. A recombinant viral nucleic acid comprising the polynucleotide according to  claim 1 . 
     
     
       17. A recombinant virus comprising the recombinant viral nucleic acid according to  claim 16 . 
     
     
       18. A plant cell infected with a recombinant virus according to  claim 17 . 
     
     
       19. The plant cell according to  claim 18  wherein the plant cell is a monocot plant cell. 
     
     
       20. A plant infected with a recombinant virus according to  claim 17 . 
     
     
       21. The plant according to  claim 20  wherein the plant is a monocot plant cell. 
     
     
       22. A polynucleotide according to  claim 1  wherein the protein of interest is a viral protein. 
     
     
       23. A recombinant viral nucleic acid comprising the polynucleotide according to  claim 22 . 
     
     
       24. A recombinant virus comprising the recombinant viral nucleic acid according to  claim 23 . 
     
     
       25. A viral genome comprising at least one duplicated genomic nucleic acid component, wherein the duplicated genomic nucleic acid component encodes a promoter operatively linked to a fusion protein, wherein the fusion protein comprises 
       (a) a viral protein obtained from a plant single-stranded RNA virus, wherein the plant single-stranded RNA virus is a barley stripe mosaic virus (BSMV),  
       (b) a protein of interest, and  
       (c) an autoproteolytic peptide comprising no more than 20 amino acids,  
       wherein the autoproteolytic peptide comprises a 2A autoproteolytic peptide from a foot and mouth disease virus (FMDV), wherein (c) is fused between (a) and (b).